ABSTRACT The premise of this work is based on the accumulated evidence that mitochondrial remodeling takes place in the stressed heart after ischemic injury, and occurs not only in the area at risk, but also in the remaining viable myocardium that must now alter its work capacity. We previously discovered that ischemia and reperfusion resulted in mitophagy and biogenesis regulated at the level of RNA translation. This application will explore this finding further by examining the dynamic protein composition of the active polysomes in hearts subjected to ischemic stress. Aim 1 will test the hypothesis that regulatory molecules associate with polysomes during ischemic stress and, in doing so, select mitochondria-targeted mRNAs for active translation. It will further determine if miRNAs, or rather the loss of certain miRNAs, also govern this process. Results from this project will develop a detailed portrait of the newly-synthesized proteins in the setting of recovery from ischemia, using metabolic labeling with a mass spectrometry-compatible Met analog (azidohomoalanine, AHA) to define the new protein proteome. Work in Aim 2 will define the relationship between alterations in mitochondrial function and changes in the metabolome that alter protein acetylation and transcriptional output. Work in this aim will employ various interventions to perturb glycolysis and oxidative phosphorylation, characterize the metabolome, and correlate results with specific alterations in protein acetylation and the transcriptome, including both mRNA and miRNA. Experiments will also be performed to explore perturbations, including a high-fat diet and variations in mitochondrial haplogroup [using cybrid cells and the mitochondrial nuclear exchange (MNX) mouse lines]. The multi-omics analysis will require the development of novel bioinformatics tools to identify relationships and control nodes mediated through changes in metabolite levels. We have already identified a common pathway shared by pioglitazone and GLP1Ra which increase the NADH/NAD+ ratio, inactivate sirtuins, result in increased protein acetylation, and lower the expression and activity of miR-33. These studies will reveal the mechanisms governing translational control of mitochondrial biogenesis, which may lead to the development of new therapeutic tools for regulating this process in the setting of ischemia/reperfusion injury. The second and third aims will yield a new understanding of the role that metabolism and mitochondrial output has on transcription, and is expected to identify metabolites of particular importance in transcriptional regulation that may govern cardiac remodeling. This project is focused on mechanisms of post-infarction remodeling and will yield a new understanding of the link between mitochondrial function, metabolic output, and transcriptional regulation in the surviving myocardium after infarction.